Effect of Phenylmethylsulfonyl Fluoride on Sterol Biosynthesis in 10,000 X g Supernatant Fraction of Rat Liver Homogenates”

Phenylmethylsulfonyl fluoride (PMSF), a reagent commonly employed for the inhibition of serine proteases, has been found to cause significant inhibition of the incorporation of labeled acetate, but not mevalonate, into nonsaponifiable lipid and digitonin-precipitable sterols in the 10,000 X g supernatant fraction of rat liver homogenate preparations. In two experiments, the extent of inhibition of the synthesis of digitonin-precipitable sterols from acetate by PMSF at 1 mM was 81 and 65%. PMSF inhibited the synthesis of nonsaponifiable lipid from acetate at concentrations as low as 0.1 microM. Preincubation of the 10,000 X g supernatant fraction of rat liver homogenates with PMSF (1 mM) resulted in a significant reduction of the activities of acetate thiokinase and 3-hydroxy-3-methylglutaric acid (HMG)-CoA synthase, but did not affect the activities of acetoacetyl-CoA thiolase. Preincubation of rat liver microsomes with PMSF (1 mM) caused a 50% reduction in the level of HMG-CoA reductase activity. The combined results indicate that major sites of action of PMSF in the inhibition of sterol biosynthesis from labeled acetate appear to be on the activities of acetate thiokinase, HMG-CoA synthase, and HMG-CoA reductase. Another reagent used to inhibit serine proteases, diisopropylfluorophosphate, had (at a concentration of 1 mM) no effect on the activities of cytosolic acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase.

In the course of studies relative to the mechanism(s) of action of selected oxygenated sterol inhibitors of cholesterol biosynthesis, we wished to explore the possibility that the effect of these oxygenated sterols was due to an effect on the proteolytic degradation of HMG'-CoA reductase or some other enzymes or processes involved in the biosynthesis of cholesterol. Accordingly, we initiated studies with phenylmethylsulfonyl fluoride, an inhibitor of serine proteases (1-4), to explore these possibilities. To our surprise, PMSF itself was found to be a relatively potent inhibitor of the synthesis of digitonin-precipitable sterols from acetate, but not from mevalonate, in the 10,000 X g supernatant fraction of rat liver * This research was supported in part by Grants HL-15376 and HL-22532 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
#' Recipient of a predoctoral fellowship from the National Science homogenates. Moreover, we wish to report the effects of PMSF on the activities of the enzymes involved in the biosynthesis of mevalonate in in vitro preparations of rat liver.

DISCUSSION
We wished to explore the possibility that the inhibitory action of some new inhibitors of cholesterol biosynthesis might be due to an effect on the proteolytic degradation of one or more proteins involved in the biosynthesis of cholesterol or in the regulation of this process. Sogawa and Takahashi (22) recently reported the presence of a serine protease in the microsomal fraction of rat liver. We therefore initiated studies of the effects of inhibitors of serine proteases to explore these possibilities. Two commonly used reagents for this purpose are DIFP and PMSF. The former compound has been shown to inhibit a variety of serine esterases and proteases such as subtilisin (23), chymotrypsin (24, 25), trypsin (25), thrombin (26), and plasmin (27). Detailed studies have shown that the modification by this reagent is specific for a particular serine residue at the catalytic site of these enzymes (23, 24, 26-29). Moreover, DIFP has been shown to have no effect on several thiol-specific proteases, including cathepsin B (30). Thus, DIFP appears to be a notably specific reagent for the mod& cation of catalytically active serine residues in proteases of this type. However, its toxicity and physical properties (a volatile liquid) have led to its replacement by other, more convenient reagents in many applications for this purpose. PMSF is the most commonly employed replacement compound for DIFP. It has been reported to be equivalent in its action to DIFP on a variety of serine proteases and esterases (3, 26, 31, 32). Accordingly, our initial studies employed PMSF.
In early phases of the present study, we found that PMSF caused an inhibition of the synthesis of digitonin-precipitable sterols from labeled acetate in the 10,000 X g supernatant fraction of rat liver homogenate preparations. The results of subsequent studies showed that as little as 0.1 PM PMSF caused significant inhibition of the synthesis of nonsaponifiable lipids from labeled acetate in the same in vitro system. The finding that PMSF, at 1 mM, caused a marked inhibition of the synthesis of nonsaponifiable lipids and digitonin-precipitable sterols from labeled acetate, but not from labeled MVA,

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suggested that a major site of the inhibitory action of PMSF was on one or more of the reactions involved in the formation of MVA from acetate. In view of this finding, we investigated the effect of PMSF on the activity of each of the enzymes involved in the conversion of acetate to MVA. As noted above, at a concentration of 1 mM, PMSF was found to cause significant reduction of the apparent activities of three of the four enzymes involved in the formation of MVA from acetate.
Acetate thiokinase, which catalyzes the formation of acetyl-CoA in an ATP-dependent reaction (33), occurs in both the mitochondrial and supernatant fractions of rat liver (34). The mitochondrial enzyme from ox heart has been extensively purified (35) and has been found to contain eight sulfhydryl groups/mol of enzyme. Londesborough et al. (35) have reported that p-hydroxymercuribenzoate reacts rapidly at 20 "C with four of the thiol groups of the enzyme, resulting in a rapid inhibition of the enzyme which could be reversed by either CoA or mercaptoethanol. These workers suggested that this inhibition may be due to an alteration of the structure of the enzyme rather than indicating that a thiol residue was playing a direct role in the catalysis by the enzyme. In the present study, preincubation of the 10,000 x g supernatant fraction of rat liver homogenate preparations with either of two thiol reagents, N-ethylmaleimide and sodium iodoacetate, at 1 mM, caused a marked reduction of the activity of acetate thiokinase. Preincubation of the same enzyme preparation with PMSF (1 mM) was also found to cause a very substantial reduction of the activity of acetate thiokinase under the conditions studied.
Acetoacetyl-CoA thiolase has been shown to exist not only in the mitochondria, wherein it participates in fatty acid oxidation and ketogenesis, but also in the cytosol (36-40). Cytosolic acetoacetyl-CoA thiolase catalyzes the formation of acetoacetyl-CoA required for the synthesis of HMG-CoA which is required for the formation of mevalonic acid and products derived therefrom, including cholesterol. The cytosolic enzyme from avian liver has been purified to homogeneity (39). The activity of this form of the enzyme has been shown to be affected by cholesterol feeding (39-41), fasting (40), and cholestyramine feeding (41) and has been reported to be very sensitive to the concentration of CoA (39). In the present study preincubation of the 10,000 x g supernatant fraction of rat liver homogenates with sodium iodoacetate (1 mM) had no effect on acetoacetyl-CoA thiolase activity. Similarly, preincubation of the enzyme preparation with PMSF (1 mM) or DIFP (1 mM) had no effect on thiolase activity.
Liver and kidney contain a mitochondrial HMG-CoA synthase which has a functional role in ketogenesis (40, 41). A separate cytosolic HMG-CoA synthase, reported to be present in all avian tissues, catalyzes the formation of HMG-CoA required for cholesterol biosynthesis (40,41). Cytosolic HMG-CoA synthase activity of liver is reduced upon cholesterol feeding or starvation and is increased upon cholestyramine administration (41). Lane and his associates have provided clear evidence for an intimate role of a cysteinyl sulfhydryl group in the catalytic activity of the liver enzyme (42). In the present study, preincubation of sodium iodoacetate (1 m~) and of PMSF (1 mM) with the 10,000 X g supernatant fraction of rat liver homogenate preparations resulted in a significant reduction of the activity of HMG-CoA synthase activity. However, preincubation with DIFP (1 mM) was without effect on the synthase activity.
The inhibition of the activity of HMG-CoA reductase by phydroxymercuribenzoate and iodoacetamide has been reported (43-46). In the present study, preincubation of PMSF (1 and 3 mM) with rat liver microsomes caused a marked reduction in the activity of HMG-CoA reductase. Preincuba-tion of the microsomal preparation with DIFP (1 mM) had no effect on HMG-CoA reductase activity. The activity of HMG-CoA reductase is subject to control by a variety of processes both in vivo and in vitro. One of these controls is the postulated kinase-phosphatase system responsible for the phosphorylation and dephosphorylation of the enzyme (13-21). Under the conditions employed in this study, the EDTA present in the HMG-CoA reductase assay buffer ensures that Mg2+-dependent kinases are inactive. However, the phosphatase has been reported to be active during the liver homogenate preparation and preincubation and has been reported to cause an overall increase in the apparent activity of HMG-CoA reductase due to conversion of the inactive phosphorylated form to the active nonphosphorylated form (14-17). It is clear that a possible site of action of PMSF could be inactivation of the phosphatase, which would give an apparent decrease in HMG-CoA reductase activity. To investigate this possibility, the effect of PMSF was tested on microsomal preparations which were isolated, preincubated, and assayed in the presence of 50 mM NaF, conditions which have been reported to inhibit the reductase-phosphatase activity (14,17). Under these conditions, PMSF, at a concentration of 1 mM, still caused a significant inhibition of HMG-CoA reductase activity (Table VI). As a further control, the effect of 1 mM NaF on HMG-CoA reductase activity was investigated to determine whether or not a complete hydrolysis of the PMSF to a-toluene sulfonate and fluoride would generate enough fluoride ion to inactivate the reductase-phosphatase sufiiciently to account for the observed inhibitions. Under the conditions employed, NaF (1 mM) was not an effective inhibitor of apparent HMG-CoA reductase activity. Taken together, these data imply that the effect of PMSF, under the conditions employed, is an inhibition of HMG-CoA reductase activity rather than an inhibition of the reductase-phosphatase activating system.
The combined results of these experiments suggest that the inhibition of sterol biosynthesis from labeled acetate in the 10,000 X g supernatant fraction of rat liver homogenates is not due to an effect of PMSF on a serine protease but by reduction of the levels of activity of acetyl-coA synthetase, HMG-CoA synthase, and HMG-CoA reductase. It is worthy to note that these enzymes have been proposed to have an essential thiol involved in the catalysis and/or have been shown to be inhibited by sulfhydryl reagents. While it is tempting to ascribe the inhibitory action of PMSF on these enzymes to the reaction of PMSF with one or more thiol groups on the enzyme (vide infra), further studies will clearly be required to explore this possibility.
The results of the experiments described herein indicate that the differences in specificity between PMSF and DIFP may be greater than commonly assumed. We have found that PMSF, at a concentration of 1 mM, causes a reduction in the levels of activity of several enzymes which are considered to be thiol-dependent enzymes, particularly HMG-CoA synthase and HMG-CoA reductase. DIFP, at the same Concentration, had no effect on the activities of these two enzymes. Thus, it appears that PMSF-induced inhibition of the activities of HMG-CoA synthase and HMG-CoA reductase is due to some mechanism other than reaction with a serine residue at or near the active site of these enzymes. Indeed, since the inhibitions by sodium iodoacetate are very similar to those caused by PMSF, it is tempting to speculate that the sulfonyl fluoride may be reacting with an essential thiol residue in these enzymes. However, other active nucleophiles, such as lysine, histidine, or arginine, might be expected to react with PMSF under the appropriate conditions; therefore, a definitive statement on the exact mode of action of PMSF on these enzymes is premature. However, it is clear that PMSF, at a concentration of 1 mM, causes inhibitions of the activities of HMG-CoA synthase and HMG-CoA reductase while these activities are unaffected by DIFP at the same concentration. These fiidings indicate that it is unlikely that the inhibitory action of PMSF is due to its commonly assumed action at a serine residue. It is important to note that several other cases of the inhibition by PMSF of enzymes other than serine proteases have been reported. Whitaker and Perez-Villasenor (47) have reported that PMSF causes a marked inhibition of papain which was accompanied by a complete disappearance of the free sulfhydryl group of the one cysteine residue of this enzyme. Kumar (48) has reported that purified fatty acid synthetase complex of pigeon liver is inhibited by PMSF. Only the palmityl-CoA deacylase component of the complex was inhibited by PMSF. The inactivation by PMSF was reversed by treatment with dithiothreitol. Recently, Inoue et al. (49) have reported that PMSF causes an inactivation of y-glutamyl transpeptidase. Sekar and Hageman (50) have recently reported that treatment of the 18,000 X g supernatant fraction of rabbit liver with [3H]phenylmethylsulfonyl fluoride led to the formation of at least 15 labeled proteins which could be detected upon electrophoresis on isoelectric focusing gels or on detergent gels.
On the basis of the results cited above, coupled with the results presented in the current study, we fully agree with the conclusion of Sekar and Hageman that inferences regarding protease function based upon treatment of complex systems or whole cells with PMSF should be made with caution. In addition, the combined results certainly also raise caution with respect to possible undesired reactions in the common practice of employing PMSF to inhibit enzymatic proteolysis during protein isolation and purification. It is worthy to note that PMSF (1 mM) has been employed in early stages of the isolation of cytosolic HMG-CoA synthase from chicken liver (11). However, no apparent major loss of activity of the enzyme was observed under the conditions employed.